Glucose-6-phosphate dehydrogenase plays critical role in artemisinin production of <Emphasis Type="Italic">Artemisia annua</Emphasis> under salt stress

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H₂O₂ and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H₂O₂ and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H₂O₂ and NO production in A. annua seedlings under the salt stress. The induced H₂O₂ was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H₂O₂ production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.     

Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

High-Level Production of Amorpha-4,11-Diene,a Precursor of the Antimalarial Agent Artemisinin,in Escherichia coli

Background

Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.

Methodology/ Principal Findings

By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.

Conclusions/ Significance

Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.     

在大肠杆菌内引入甲羟戊酸途径高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯

以青蒿素为基础的联合药物疗法 (ACTs) 被认为是目前治疗恶性疟疾的最有效方法。然而青蒿素供应不足且价格昂贵,限制了ACTs的广泛使用。采用基因工程手段构建异源类异戊二烯生物合成途径,利用大肠杆菌发酵能高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯。首先在大肠杆菌Escherichia coli DHGT7中引入人工合成的紫穗槐-4,11-二烯合酶基因,利用大肠杆菌内源的法尼基焦磷酸,成功获得了紫穗槐-4,11-二烯。为提高前体供给,引入粪肠球菌的甲羟戊酸途径,紫穗槐-4,11-二烯的产量提高了13     

Nicotiana benthamiana as a production platform for artemisinin precursors

Background

Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment.

Methodology/Principal Findings

Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg⁻¹ fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana.

Conclusion/Significance

This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Δ11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1α was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.     

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Abstract

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.     

The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua.     

Exogenous salicylic acid-mediated modulation of arsenic stress tolerance with enhanced accumulation of secondary metabolites and improved size of glandular trichomes in <Emphasis Type="Italic">Artemisia annua</Emphasis> L<Emphasis Type="Italic">.</Emphasis>

The present study was undertaken to find out individual and interactive effects of arsenic (As) and salicylic acid (SA) on an important medicinal plant, Artemisia annua. As uptake and its accumulation was detected and found to be maximum in roots at higher As concentration (150 μM). Under As treatments, H₂O₂ and MDA content were induced. Biomass and chlorophyll content were negatively affected under As treatments. Furthermore, enzymatic (SOD, CAT, APX, and GR) and non-enzymatic antioxidants were also enhanced under As treatments. Exogenous application of SA reduced the extent of H₂O₂ and O₂ ^? generation and lipid peroxidation, while reverted biomass and chlorophyll content to overcome oxidative stress. Simultaneous application of SA with As increased endogenous SA level, artemisinin, and dihydroartemisinic acid as compared with individual As treatment and pre-application of SA with As treatments. The expression of four key artemisinin biosynthetic pathway genes, i.e., ADS, CYP71AV1, DBR2, and ALDH1 were upregulated at a maximum in plants simultaneously treated with SA and As. Similar pattern of artemisinin accumulation and glandular trichome size was observed which attest that SA has a stimulatory impact on artemisinin biosynthesis under As stress. Our study suggests that exogenous application of SA and As together induced more tolerance in A. annua than a comparable dose of SA pre-treatment. The study may provide a platform with dual benefits by developing As-tolerant plants to be used for phytoremediation of arsenic from As-contaminated soil and obtaining high artemisinin-producing A. annua plants.     

From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (<Emphasis Type="Italic">Coffea arabica</Emphasis> L. × <Emphasis Type="Italic">Coffea canephora</Emphasis> Pierre ex A. Froehner)

Gibberellins (GAs) are diterpenoid hormones, control various physiological developments in plants. The role of gibberellins on morphology and secondary metabolite production was examined in Artemisia annua, a medicinal plant that has been acknowledged as a source of artemisinin, an antimalarial compound. Subsequently, the GA20ox gene from Torenia fournieri (TfGA20ox2) was transferred to A. annua by Agrobacterium-mediated transformation. Compared with wild type plants, all nine transgenic plants showed significantly higher plant heights and artemisinin contents. The highest artemisinin content and yield in TfGA20ox2-overexpressing plants was around two-fold higher than wild type. Moreover, transgenic plants had higher numbers of branches (52.4%) and greater branch lengths (60–203%), but smaller leaf size (77.6%). Interestingly, relative to wild type the number and size of glandular trichomes in transgenic leaves was about 30 and 35% higher, respectively. From GC–MS analysis, the proportion of diterpenes in transgenic plant extracts was 1.5-fold lower than those noticed in wild type, while the proportion of sesquiterpenes was increased about 1.6 times when compared to wild type. However, the content proportion of monoterpenes showed a slightly increase, whereas the level of triterpenes showed no variation. In addition, two monoterpenes (eucalyptol and borneol), four sesquiterpenes (α-caryophyllene, β-guaiene, δ-cadinene and β-cubebene) and one triterpenes (isomultiflorenone) were detected only in transgenic extract, whereas d-α-tocopherol, a diterpenoid compound was found only in wild type but not transgenic plant. These results suggested that gibberellins play a significant role in regards to morphology, trichome formation and terpenoid metabolite production in A. annua.     

Salicylic acid activates artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.     

Cumulative role of bioinoculants on growth,antioxidant potential and artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> L. under organic field conditions

Artemisia annua L. is mostly known for a bioactive metabolite, artemisinin, an effective sesquiterpene lactone used against malaria without any reputed cases of resistance. In this experiment, bioinoculants viz., Streptomyces sp. MTN14, Bacillus megaterium MTN2RP and Trichoderma harzianum Thu were applied as growth promoting substances to exploit full genetic potential of crops in terms of growth, yield, nutrient uptake and particularly artemisinin content. Further, multi-use of the bioinoculants singly and in combinations for the enhancement of antioxidant potential and therapeutic value was also undertaken which to our knowledge has never been investigated in context with microbial application. The results demonstrated that a significant (P < 0.05) increase in growth, nutrient uptake, total phenolic, flavonoid, free radical scavenging activity, ferric reducing antioxidant power, reducing power and total antioxidant capacity were observed in the A. annua treated with a combination of bioinoculants in comparison to control. Most importantly, an increase in artemisinin content and yield by 34 and 72 % respectively in the treatment having all the three microbes was observed. These results were further authenticated by the PCA analysis which showed positive correlation between plant macronutrients and antioxidant content with plant growth and artemisinin yield of A. annua. The present study thus highlights a possible new application of compatible bioinoculants for enhancing the growth along with antioxidant and therapeutic value of A. annua.     

Artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> and metabolic engineering: questions,challenges, and perspectives

Artemisinin-based combination therapy (ACT) forms the frontline treatment of malaria. Artemisinin, an endoperoxide sesquiterpenoid lactone biosynthesized by Artemisia annua, is the effective medicine that kills malarial parasites. Due to insufficient production of artemisinin for ACT, millions of people lost their lives in past years worldwide. To solve this severe problem, numerous studies have been undertaken to understand artemisinin biosynthesis and to innovate metabolic engineering technology to increase artemisinin yield. Here, we focus on reviewing progresses achieved in understanding biosynthetic pathway, genetic breeding, metabolic engineering, and synthetic biology. Furthermore, based on current knowledge, we discuss multiple fundamental questions and challenges.     

Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis

The endoperoxide sesquiterpene lactone artemisinin and its derivatives are a promising new group of drugs against malaria. Artemisinin is a constituent of the annual herb Artemisia annua L. So far only the later steps in artemisinin biosynthesis--from artemisinic acid--have been elucidated and the expected olefinic sesquiterpene intermediate has never been demonstrated. In pentane extracts of A. annua leaves we detected a sesquiterpene with the mass spectrum of amorpha-4,11-diene. Synthesis of amorpha-4,11-diene from artemisinic acid confirmed the identity. In addition we identified several sesquiterpene synthases of which one of the major activities catalysed the formation of amorpha-4,11-diene from farnesyl diphosphate. This enzyme was partially purified and shows the typical characteristics of sesquiterpene synthases, such as a broad pH optimum around 6.5-7.0, a molecular mass of 56 kDa, and a K(m) of 0.6 microM. The structure and configuration of amorpha-4,11-diene, its low content in A. annua and the high activity of amorpha-4,11-diene synthase all support that amorpha-4,11-diene is the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin.